A Network Pharmacology Approach To Discover Active Compounds Combination And Its Mechanisms Of Xiaoyaosan On Anti-liver Fibrosis

Purpose:In order to predict and analyze the effective compounds combination（LLAAF,Luteolin+Licochalcone a+Aloe-emodin+Acacetin formula）from Xiaoyaosan and their mechanism,we used network pharmacology and transcriptomics.Methods:（1）The extract of XYS was analyzed HPLC-MS to make a quality control;male Wistar rats were intraperitoneally injected with 50%CCl₄ solution at 1 ml/kg twice a week,all rats in drug-treated group was administered with XYS from 5 weeks;after 9 weeks,the liver index,histopathology,serum liver function tests,liver fibrosis tests,liver tissue hydroxyproline（Hyp）,collagen I expression and?SMA protein expression were assayed.（2）The major XYS treatment network was structured by network topology parameter 5 principle method;the network contribution scores were generated by entropy method and weighted summation method;the network contribution scores are evaluated for screening compound combinations and sorted.（3）Compounds and compounds combinations in L02,T6 and Lx2 cells were screened using MTS method;the effect of LLAAF on type I collagen and?SMA protein expression in T6 and Lx2 cells were detected;the histopathology,liver tissue Hyp,collagen I expression and?SMA protein expression in rats were detected.（4）The differential lnc RNA and m RNA and lnc RNA-related transcription factors were chosen to the bioinformatics network background;The core sub-network composition analysis,compound function and contribution prediction were carried out;p-Fox O3a/Fox O3a,p-STAT3/STAT3,p-Akt/Akt,p-Smad3/Smad3 ratios and c-Myc protein expression in Lx2 cells and rat liver tissue were determined.Results:（1）Compared to the model group,XYS treatment improved histopathological changes,liver index,serum ALT,AST,TBIL,HA,LN,PC-III,IV-C,liver tissue Hyp,protein expression of?SMA and Collagen I（P<0.05）.（2）8 key compounds were found.255 potential compound combinations were evaluated by network contribution scores.（3）LLAAF inhibited the activity of T6 and Lx2 cells,and the effective concentration was lower than the its concentration in L02 cells;compared to the model group,LLAAF treatment can significantly reduce the expression of?SMA and Collagen I protein in Lx2 and T6 cells（P<0.05）;compared to the model group,LLAAF and XYS could improve the steatosis and collagen fiber deposition,reduce the expression of?SMA and Collagen I protein and Hyp content（P<0.05）.（4）Compared with the XYS and LLAAF network,the shared pathway including Jak-STAT and Fox O signaling pathways and c-Myc target protein were obtained;The core subnetwork a of the LLAAF is mainly related to the PI3K-Akt signaling pathway and the Fox O signaling pathway,the core subnetwork b affects both the Notch and Wnt signaling pathway,and the core subnetworks c and d affect the Jak-STAT signaling pathway;In cells and rat liver tissues,compared to the model group,LLAAF treatment reduced p-Fox O3a/Fox O3a,p-STAT3/STAT3,p-Akt/Akt,p-Smad3/Smad3 ratio,and c-Myc protein expression（P<0.05）.Conclusion:The LLAAF from XYS can anti-liver fibrosis;its efficacy is related to synergistic effect of four compounds;the LLAAF has better efficacy;the anti-fibrosis effect of LLAAF may be related to the down-regulation of Jak-STAT,PI3K-Akt-Fox O signaling pathway and c-Myc protein expression,which in inhibits HSC activation.It may be able to play a certain role in the clinical use and drug development of XYS.